Microfluidic Electrochemical Device and Process for Chemical Imaging and Electrochemical Analysis at the Electrode-Liquid Interface In-Situ

ABSTRACT

A microfluidic electrochemical device and process are detailed that provide chemical imaging and electrochemical analysis under vacuum at the surface of the electrode-sample or electrode-liquid interface in-situ. The electrochemical device allows investigation of various surface layers including diffuse layers at selected depths populated with, e.g., adsorbed molecules in which chemical transformation in electrolyte solutions occurs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part of U.S. patent applicationSer. No. 13/047,025 filed 14 Mar. 2011 entitled “Systems and Methods forAnalyzing Liquids under Vacuum”, now issued as U.S. Pat. No. 8,555,710,which reference is incorporated herein in its entirety.

STATEMENT REGARDING RIGHTS TO INVENTION MADE UNDER FEDERALLY-SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under ContractDE-ACO5-76RL01830 awarded by the U.S. Department of Energy. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to electrochemical devices andprocesses. More particularly, the invention relates to a microfluidicelectrochemical device and process for chemical imaging andelectrochemical analyses of analytes at the electrode-liquid interfacein-situ.

BACKGROUND OF THE INVENTION

The electrode-liquid interface is the most common interface encounteredin electrochemical systems and is of great and diverse technologicalimportance. At the molecular level, surface atoms have a differentchemical environment than those present in the bulk environment. Thus, adirect observation and fundamental understanding of charge transport,phase transitions, growth of solid interfaces (e.g., adlayers), andreactivity at the electrode-electrolyte interface are needed. The term“adlayer” refers to adsorbed chemical species that form layers on thesurface of the electrode and chemically interact with the electrode orsubstrate. While surface science techniques have made investigation ofadsorbed molecules possible, molecular-scale surface science studies areconducted primarily at solid-gas interfaces and solid-vacuum interfacesdue to challenges associated with applying surface-sensitive vacuumtechniques to high volatility liquids. Consequently, detailed in-situstudies at the electrode-solution interface using surface-sensitivetechniques are especially lacking. Ex-situ electrochemical experimentsinvolve removing an electrode from an electrolyte and analyzing sampleadlayers present on the electrode in an ambient atmosphere or in anultra-high vacuum (UHV) instrument or system. However, evenwell-characterized emersion adlayers may only partially or incompletelyrepresent the real in-situ system. Further, in-situ chemical imaging ofactual electrode-electrolyte interfaces has not yet been achieved. Thus,there remains a need for in-situ measurements of electrochemical systemsthat probe the electrode-liquid sample or liquid interface rather thanrelying on ex-situ analyses. In addition, new chemical imagingapproaches are needed that employ vacuum-based techniques suitable forstudy of surfaces of high-vapor-pressure liquids and electrode-solutioninterfaces. The present invention addresses these needs.

SUMMARY OF THE INVENTION

A microfluidic electrochemical device and process are detailed forchemical imaging and electrochemical analysis of analytes at theelectrode-liquid sample interface in-situ. The present inventionincorporates a microfluidic device for analyzing fluids in a vacuumdetailed, e.g., in U.S. patent application Ser. No. 13/047,025 filed 14Mar. 2011, now allowed. The present invention provides simultaneousmultimodal analyses in concert with combined electrochemical analysisand chemical imaging in real-time or as a function of time thatelucidates spatial distributions of various analytes as reactions occurin the liquid sample. The microfluidic electrochemical device mayinclude an electrochemical microfluidic flow chamber (cell) that definesa liquid flow path through the flow chamber. The flow chamber of themicrofluidic electrochemical device may be positioned on a siliconsubstrate such as a silicon wafer or a silicon chip. The electrochemicalflow chamber may include one or more inlets and one or more outlets thatdeliver liquid samples to and from the electrochemical flow chamber,respectively. In some applications, the inlets and the outlets includeone or more branches. In some applications, the inlets and the outletsmay be positioned apart from another inlet or outlet or include aselected separation distance. The electrochemical flow chamber mayinclude a flow channel with a selected depth. In various applications,the flow chamber may include a flow channel depth between about 0.1 μmand about 1000 μm or greater. In some applications, the flow chamber mayinclude a flow channel depth between about 1 μm and about 1000 μm orgreater. The flow chamber may include a support membrane with one ormore detection apertures. The support membrane may be constructed of, orinclude, a material such as silicon nitride (SiN), silicon dioxide(SiO₂), including combinations of these various materials.

Probe beams may be delivered from selected analytical instrumentsthrough the detection aperture(s) under vacuum into the flow chamberthat exposes surfaces of liquid (e.g., liquid) samples including, e.g.,liquids, solutions, biological broths, cell growth media containing oneor more analytes to the probe beams to provide chemical imaging analysisof analytes at surfaces of liquid samples when the liquid samples areintroduced past the detection aperture(s). Surface-sensitive analyticalinstruments for chemical imaging of liquid sample analytes include,e.g., X-ray photoelectron spectroscopy (XPS); scanning electronmicroscopy (SEM); transmission electron microscopy (TEM); time-of-flightsecondary ion mass spectrometer (ToF-SIMS); helium ion microscopy(HeIM); Auger electron spectroscopy (AES); and Rutherford backscatteringspectrometry (RBS). Probe beams from analytical instruments may beintroduced into the liquid samples at selected depths, layers,locations, and areas (e.g., within a few microns) to analyze analytes.In various applications, surfaces of liquid samples and workingelectrode/liquid sample interfaces may be probed with probe beams fromselected analytical instrument(s) to selected depths. For example, probebeams from ToF-SIMS instruments may interrogate surfaces to a depth ofabout 6 nm. However, no depth limitations are intended by the exemplaryinstrument. In some applications, depth of probe beams may be selectedthat permit selected regions or layers of the electrode/liquid sampleinterface to be probed. For example, at selected depths, adsorbedmolecules, monolayer films (e.g., 2 Å to 10 Å), diffuse-layers (1 nm to1 μm), and/or modified films (e.g., 1 nm to 1 μm) may be investigated.

The microfluidic electrochemical device may also include electrodes sucha working electrode, a counter electrode, and/or a reference electrode.Electrodes provide electrochemical analyses of analytes in liquidsamples, e.g., in concert with cyclic voltammetry (CV). Electrodes inthe microfluidic electrochemical device may be in the form of wires,thin films, and sputter-deposited thin films. Electrodes may beconstructed of, or include, metals, metal oxides, carbon, graphene, orother suitable electrode materials including combinations of thesevarious materials. In some applications, the working electrode and thecounter electrode may be integrated with a reference electrode on asingle substrate. In some applications, the counter electrode and thereference electrode may be positioned on a substrate that is differentfrom the substrate that contains the working electrode. In someapplications, the working electrode may be positioned above the flowchannel within the flow chamber of the electrochemical device andpositioned beneath the detection aperture (window). The counterelectrode and the reference electrode may be disposed below the flowchannel in the flow chamber. The working electrode may be configured toapply a selected potential into the sample between the working electrodeand the reference electrode to drive reactions in the liquid sample as afunction of time, space, and/or potential. The counter electrode may beconfigured to measure electrical current stemming from reactionsinvolving analytes in the liquid sample at the surface of the workingelectrode. The microfluidic electrochemical device is configured toprovide electrochemical analysis and chemical imaging of analytes atsurfaces of the liquid sample and at the working electrode-liquidinterface in-situ individually or simultaneously at selected depths orselected layers. Electrodes in the microfluidic electrochemical devicemay be coupled to an external workstation that is configured to deliverpotentials between the working electrode and reference electrode and tomeasure current between the working electrode and the counter electrode.

The present invention also includes a process for simultaneouselectrochemical analysis and chemical imaging of analytes present insamples of various types. The process may include introducing a liquidsample containing one or more analytes through a liquid flow path in themicrofluidic flow chamber of a microfluidic electrochemical device.Selected potentials may be delivered between a working electrode and areference electrode in the microfluidic flow chamber to drive reactionsof the one or more analytes in the liquid sample as a function of time,space, and/or potential. The sample in the liquid flow path may beexposed to one or more probe beams from selected analytical instrumentsunder vacuum to provide chemical imaging of analytes at a selected depthor a selected layer of a liquid sample or at the workingelectrode-liquid sample interface in-situ.

In some applications, the method may provide simultaneous chemicalimaging and electrochemical analyses of analytes including chemical andmolecular species present at the working electrode-liquid sampleinterface in-situ including selected locations, depths, layers, andsurfaces thereof.

Electrochemical analysis may be provided, e.g., by cyclic voltammetry.Chemical imaging may be provided in concert with an analytical methodincluding, but not limited to, e.g., X-ray photoelectron spectroscopy(XPS); scanning electron microscopy (SEM); secondary ion massspectrometry (SIMS); helium ion microscopy (HeIM); Auger electronspectroscopy (AES); Rutherford backscattering spectrometry (RBS);transmission electron microscopy (TEM), and combinations of thesevarious methods.

Electrical current between the working electrode and a counter electrodemay be measured to provide electrochemical analysis of various chemicaland molecular species stemming from reactions involving variousanalytes. Various potentials may be delivered from an electrochemicalworkstation positioned external to the microfluidic electrochemicaldevice. Current may be measured by the same electrochemical workstation.In some applications, the liquid sample may include an electrolyte. Insome applications, the sample may be a buffer solution or include abuffer. In some applications, the sample may be a biological samplecontaining one or more biological analytes selected from: cells,bacteria, other biological components, and combinations of these variousbiological analytes.

In some applications, chemical imaging and electrochemical analysis mayinclude determining chemical species at the surface of the workingelectrode-liquid sample interface in-situ. In some applications,chemical imaging may include imaging adsorbed molecules at the surfaceof the working electrode and in solutions adjacent the working electrodein-situ. In some applications, the method may include observing ortracking compositional changes of an electrolyte as a function of timein-situ. In some applications, the method may include a time-resolvedand/or a space-resolved determination of reaction products andintermediate chemical species as electron transfer occurs in the samplein-situ. In some applications, the method may include electrochemicallydetermining and chemically imaging material changes to an electrode in amicrofluidic electrochemical device as potential is varied.

The purpose of the foregoing abstract is to enable the United StatesPatent and Trademark Office and the public generally, especiallyscientists, engineers, and practitioners in the art who are not familiarwith patent or legal terms or phraseology, to determine quickly from acursory inspection the nature and essence of the technical disclosure ofthe application. The abstract is neither intended to define theinvention of the application, which is measured by the claims, nor is itintended to be limiting as to the scope of the invention in any way. Amore complete appreciation of the invention will be readily obtained byreference to the following description of the accompanying drawings inwhich like numerals in different figures represent the same structuresor elements.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a side view of a microfluidic electrochemical deviceof the present invention.

FIGS. 2a-2d show different electrode configurations for the presentinvention.

FIGS. 3a-3b show two exemplary gold electrode designs for the presentinvention.

FIG. 4 is a photograph showing the microfluidic electrochemical deviceof the present invention.

FIG. 5 is a photograph showing the microfluidic electrochemical deviceof the present invention coupled to a representative chemical imaginginstrument.

FIG. 6 shows a typical cyclic voltammogram obtained with the presentinvention.

FIGS. 7a-7d show ToF-SIMS 2D chemical images of various ionic speciesacquired with the present invention.

FIG. 8 shows ToF-SIMS m/z spectra acquired with the present invention atvarious voltages.

FIG. 9 shows another configuration of the present invention for analysisof biofilms.

FIGS. 10a-10c present chemical imaging data for an exemplary biofilmgrown and analyzed with the present invention.

FIGS. 11a-11c present chemical imaging data from analysis of biofilmsacquired with the present invention.

DETAILED DESCRIPTION

A new microfluidic electrochemical device and process are detailed thatprovide combined chemical imaging and electrochemical analysis ofanalytes at the electrode-solution interface in-situ. Chemical imagingis described herein in concert with one exemplary surface sensitiveinstrument, i.e., a ToF-SIMS. However, the invention is not limitedthereto, as detailed further herein. The following description includesa best mode of the present invention. Various advantages and novelfeatures of the present invention are described herein and will becomefurther readily apparent to those skilled in this art from the followingdetailed description. As will be realized, the invention is capable ofmodification in various respects without departing from the invention.It should be understood that there is no intention to limit theinvention to the specific forms disclosed, but, on the contrary, theinvention covers all modifications, alternative constructions, andequivalents falling within the spirit and scope of the invention asdefined in the claims. Accordingly, the drawings and description of thepreferred embodiment set forth hereafter are to be regarded asillustrative in nature, and not as restrictive.

FIG. 1 is a schematic showing a side view of a microfluidicelectrochemical device 100 of the present invention for combinedchemical imaging and electrochemical analysis of analytes at theelectrode-liquid sample interface in-situ. Electrochemical device 100may include a microfluidic electrochemical flow chamber (cell) 2 with amicrofluidic flow channel 4, one or more fluid inlets 6, and one or morefluid outlets 8. The term “microfluidic” refers to the depth of flowchannel 4 in electrochemical flow chamber 2. Flow channel 4 may includea depth that varies depending on the selected application. In someembodiments, flow channel 4 may include a depth dimension greater thanor equal to about 10 micrometers (μm). In some embodiments, flow channel4 may include a depth dimension between about 10 micrometers (μm) andabout 300 micrometers (μm). In some embodiments, flow channel 4 mayinclude a depth dimension at or below 300 micrometers (μm). In theexemplary embodiment, flow channel 4 may include selected dimensions:2.5 mm (L)×2.5 mm (W)×0.3 mm (H).

One or more fluid reservoirs (not shown) may be positioned, e.g., belowflow chamber 2 at respective ends of inlets 6 and outlets 8 to containsamples that are flowed into and out of flow chamber 2. In someembodiments, fluid reservoirs may include a fluid volume or capacity ofapproximately 20 μL, but volumes are not limited. Flow channel 4 canconnect fluid inlets 6 to fluid outlets 8 to enclose the circulationloop. Both static and dynamic flow modes may be employed. Variousliquids and liquid samples may be circulated through flow chamber (cell)2 to conduct chemical imaging and electrochemical analysis. In someembodiments, an external syringe pump (described further herein) may beused to introduce selected samples into microfluidic chamber 2. In someembodiments, samples may be introduced through flow chamber 2 at a flowrate below 4 μL per min (i.e., 4 μL/min). However, flow rates are notlimited.

Electrochemical flow chamber (cell) 2 may include a top frame 12constructed of, e.g., a semiconductor wafer composed of a suitable framematerial such as silicon (Si), silicon nitride (SiN), silicon dioxide(SiO₂), and combinations of these materials. Top frame 12 may include aselected thickness. In some embodiments, top frame 12 may include athickness dimension of between about 100 μm and about 500 μm. In someembodiments, top frame 10 may include dimensions of 7.5 mm (W)×7.5 mm(L) for a coverage area of about 56.3 mm², but dimensions are notintended to be limited.

Top frame 12 may include a detection area 14 that is open to atmosphereand may include selected shapes. In the figure, detection area 14 is inthe form of a V-shaped well positioned, e.g., through the center of topframe 12, but position and shape are not limited.

Top frame 12 may include a support membrane 16 constructed of a suitablesupport material such as SiN, SiO₂, Si, or another electron transparentmaterial. In various embodiments, support membrane 16 may include athickness dimension of between about 25 nm and about 200 nm. Thicknessdepends in part on dimensions of other structural components ofelectrochemical flow chamber 2. For example, support membrane 16 mayinclude a greater thickness when top frame 12 has a greater surface areato provide suitable support. Thus, dimensions are not intended to belimited.

Support membrane 16 may be bonded to the top of microfluidic flowchamber 2 to enclose flow chamber 2. Bonding between top frame 12 (e.g.,Si wafer) and the top of microfluidic flow chamber 2 may beaccomplished, e.g., with an oxygen plasma, or another suitable bondingmethod that provides a leak-tight fluid seal in flow chamber 2.

Support membrane 16 (e.g., SiN membrane 16) may be positioned, e.g.,immediately below top frame 12 (e.g., Si frame 12) below detection area14. Support membrane 16 may include one or more detection apertures(windows) 18 that open into microfluidic flow chamber 2 to allow entryof one or more probe beams 22 from selected surface-sensitive analyticalinstruments (e.g., ToF-SIMS) into samples and solutions introduced toelectrochemical flow chamber 2 to provide chemical imaging of analytespresent in the samples. Detection apertures (windows) 18 may also bepositioned at various locations on support membrane 16, e.g., above flowchannel 4. Detection apertures (windows) 18 may be bored through supportmembrane 16 using, e.g., a primary ion beam from a ToF-SIMS instrumentor a focused ion beam from an SEM instrument or other focused energybeams. Detection apertures 18 into electrochemical flow chamber 2 may beof any size that permits entry of probe beams 22 from selectedanalytical instruments that probe liquid samples and returns analyteions in a secondary beam 24 back to the analytical instruments fordetermination or analysis. Detection apertures (windows) 18 have a sizesufficiently small to minimize mean-free path length to minimize fluidloss from microfluidic flow chamber 2 and to maximize successfulcollisions between probe beams 22 and analytes of interest when probebeams 22 are introduced into samples including, e.g., solutions andbattery electrolytes. In some embodiments, detection apertures 18 mayinclude a size less than about 3 microns (μm). In the exemplaryembodiment, detection apertures 18 may include a size of about 2 μm. Insome embodiments, detection apertures 18 may include a size less thanabout 1 micron (μm).

Prior to assembly, microfluidic electrochemical flow chamber 2 (e.g.,not yet including top frame 12 and support membrane 16) may besputter-coated with a thin (e.g., about 10 nm to about 30 nm) layer ofgold that seals pores of the elastomer on the exterior surface of flowchamber 2 to retain samples and liquids or other fluids when introducedto flow chamber 2.

Electrochemical flow chamber 2 may include up to three electrodesconfigured to perform electrochemical analyses of analytes in liquidsamples or solutions introduced to electrochemical device 100.Electrodes may include a working electrode 26, a counter electrode 28,and a reference electrode 32 that each couple to an externalelectrochemical workstation 34. Electrodes 26, 28, and 32 may beconstructed of selected metals including, e.g., gold (Au), platinum (Pt)and copper (Cu); metal oxides including, e.g., CuO, CoO, and V₂O₅; andcarbon (C) such as graphene. In the instant embodiment, workingelectrode 26 may be constructed of gold (Au) or another suitableelectrode material. Counter electrode 28 and reference electrode 32 maybe constructed of selected metals such as platinum (Pt) metal or anothersuitable electrode material.

In the instant embodiment, working electrode 26 may be positioned on anunderside of support membrane 16 at a top end of microfluidic flowchamber 2 below detection aperture 18 above flow channel 4. A metalconnecting wire 38 composed of copper or another suitable conductingmaterial of a selected length may couple (not shown) to workingelectrode 26 inside flow chamber 2. Another end of conducting metal wire38 may exit, e.g., through a side of flow chamber 2 and couple workingelectrode 26 to an electrochemical work station 34 (e.g., Model 824Electrochemical Detector, CH Instruments, Inc., Austin, Tex., USA)positioned external to flow chamber 2. Counter electrode 28 andreference electrode 32 may be positioned at a bottom end of microfluidicflow chamber 2 atop a mounting block 20 positioned below flow channel 4.Mounting block 20 may be positioned, e.g., atop an elastomer block 30detailed further herein.

Working electrode 26 may deliver a selected potential (E) into a sample(e.g., solution, electrolyte, biofilm, or other sample) between workingelectrode 26 and reference electrode 32 to drive reactions of variousanalytes in the sample as a function of time, space, and/or potential.Reference electrode 32 provides a fixed electrode potential from whichother reaction potentials may be calibrated or otherwise calculated.Counter electrode 28 may measure electrical current stemming fromreactions occurring between the various analytes in the liquid sample.Electrodes 26, 28, and 32 in combination provide electrochemicalanalysis of analytes in the liquid sample, e.g., at the workingelectrode-sample interface in-situ.

Electrochemical device 100 is configured to sustain a high vacuumcondition, e.g., achieving pressures less than about 5×10⁻⁷ Torr whenintroduced into vacuum chamber 36 of a selected analytical instrument(e.g., ToF-SIMS, SEM) during operation. Electrochemical device 100 maybe used one or more or multiple times by introducing different liquidsamples including solutions and battery electrolytes intoelectrochemical chamber (cell) 2 at various or selected conditionsincluding, e.g., ambient conditions or high vacuum conditions.Electrochemical device 100 may be discarded when performance warrants areplacement device.

Lab-on-a Chip

In some embodiments, microfluidic electrochemical device 100 may befabricated as a lab-on-a-chip device. Referring back to FIG. 1, anegative mold or template (not shown) may be used for castingmicrofluidic electrochemical chamber (cell) 2 and associated internalfeatures including, e.g., flow channel 4, inlets 6, and outlets 8 eachwith their selected dimensions. Negative molds may be fabricated on aflat silicon chip or wafer, e.g., as detailed by Yu et al. (e.g., in“Microfluid Nanofluid”, DOI 10.1007/10404-013-1199-4), which referenceis incorporated herein in its entirety. In the exemplary embodiment, thenegative mold was fabricated on silicon (Si) substrate 12. Internalfeatures were constructed using a selected photoresist material (e.g.,SU-8 photoresist, Microchem, Newton, Mass., USA) placed onto a Si wafersubstrate 12. The photoresist may be spun or spread at a selectedthickness over the silicon substrate 12. A photomask with a patternselected to provide the internal features of microfluidic flow chamber 2may be designed using design software (e.g., AutoCAD) and then printedwith a mask printer (e.g., Model SF-100 Xpress mask printer, IntelligentMicro Patterning LLC, St. Petersburg, Fla., USA). The photomask whenplaced onto Si substrate 12 and exposed to UV light transfers thedesired pattern onto the photoresist and solidifies the photoresist,creating the reverse mold on the Si substrate 12. The pattern permitscasting of microfluidic electrochemical flow chamber (cell) 2 using aselected elastomer that achieves desired high aspect ratio features andinternal (microchannel) flow structures.

In some embodiments, microfluidic electrochemical flow chamber (cell) 2may be constructed of an elastomer such as, e.g., polydimethylsiloxane(PDMS). PDMS elastomer may be mixed as a prepolymer with a curing agent(e.g., Sylgard 184, Dow Corning Co., Midland, Mich., USA), degassedunder vacuum (e.g., 30 minutes in a vacuum dessicator), and poured ontothe reverse template (mold) (not shown) located on Si substrate 12containing selected high aspect ratio features (e.g., fluid channels) ata selected thickness (e.g., 1 cm), and then cured in an oven for aselected time (e.g., ˜1 hour) at a selected temperature (e.g., 70° C. to75° C.). Cured PDMS may be removed from the template mold and cut to aselected size (˜12 mm×12 mm) to form microfluidic electrochemical flowchamber (cell) 2 with its microfluidic features including a flow channel4, one or more fluid (microchannel) inlets 6, and one or more fluid(microchannel) outlets 8 each with selected and/or respective channeldimensions detailed herein. In some embodiments, flow channel 4 mayinclude a depth of about 300 μm, but depth is not intended to belimited. In some embodiments, inlet channels 6 and outlet channels 8 mayinclude dimensions of, e.g., 100 μm (L)×100 μm (W) by 200 μm (depth).

Microfluidic electrochemical flow chamber (cell) 2 may include a baseelastomer block 30 upon which an electrode mounting block 20 may bepositioned. Electrode mounting block 20 may be constructed, e.g., ofelastomers, hard plastics, glass, silicon, including combinations ofthese various materials. In some embodiments, electrode mounting block20 may be constructed of a high-temperature epoxy (e.g., Duralco 4461epoxy, Cotronics Co., Brooklyn, N.Y., USA). Electrode mounting block 20may be formed by introducing the epoxy mixture containing a curing agentin a template patterned with SU-8 as described previously for PDMSmicrofluidic chamber 2. No limitations are intended.

In some embodiments, metal connecting wires 40 and 42 composed of copperor another suitable conducting material of a selected length may beintroduced (e.g., inserted) through two through-holes (e.g., ˜0.5 mmdiameter) (not shown) introduced e.g., with a syringe needle (e.g., aModel No.: NE-301PL-C syringe needle, Small Parts, Inc., Miramar, Fla.,USA) through a bottom end of microfluidic flow chamber 2. One end ofconnecting wires 40 and 42 introduced through the bottom end of chamber2 may be coupled respectively to counter electrode 28 (e.g., 600 μmdiameter) and to reference electrode 32 (e.g., 350 μm diameter)supported on electrode mounting block 20. Another end of conductingmetal wires 40 and 42 may exit flow chamber (cell) 2 e.g., from thebottom end to couple counter electrode 28 and reference electrode 32 toexternal electrochemical work station 34. Work station 34 may deliverselected potentials to electrodes 26, 28, and 32 of electrochemicaldevice 100.

In some embodiments, an epoxy mixture containing curing agent may beintroduced (e.g., poured) over the top of conducting metal wires 40 and42 introduced through the bottom end of microfluidic flow chamber 2 to aheight that leaves a top end of conducting metal wires 40 and 42 exposedfor subsequent coupling to counter electrode 26 and reference electrode32, respectively. The epoxy mixture may be cured for a selected time(e.g., 24 hours) at a selected temperature (e.g., room temperature) toembed conducting metal wires 40 and 42 in the epoxy of electrodemounting block 20. Cured epoxy in electrode mounting block 20 may besubsequently heated in an oven for a selected time (e.g., 2 hours) at aselected temperature (e.g., 110° C.) to anneal assembled devicecomponents. With conducting wires 40 and 42 embedded in electrodemounting block 20, surface of electrode mounting block 20 may bepolished, e.g., with fine sand papers or other polishing materials toprovide a flat surface upon which counter electrode 26 and referenceelectrode 32 may be constructed. Counter electrode 26 and referenceelectrode 32 may be constructed, e.g., by sputter-coating the desiredmetal electrode material [e.g., platinum (Pt)] onto the surface of themounting block 20 and then coupling counter electrode 26 and referenceelectrode 32 to connecting wires 40 and 42, respectively. Wires 40 and42 when coupled to counter electrode 26 and reference electrode 32 inelectrode mounting block 20 may then be connected to the electrochemicalworkstation (FIG. 1) located external to electrochemical device 100.

FIG. 2a shows a top perspective view of an exemplary electrodeconfiguration of the present invention of a thin-film design. In theinstant embodiment, counter electrode 28 and reference electrode 32 maybe thin film electrodes constructed of platinum (Pt) or another suitableelectrode material. As described previously herein, electrodes 28 and 32may be sputter-deposited electrodes (Denton Vacuum, LLC, Moorestown,N.J.). As shown in the figure, counter electrode 28 may be separatedfrom reference electrode 32 prior to sputtering with, e.g., a narrow[e.g., 3 mm (L)×0.2 mm (W)] strip of insulating tape 44 or otherinsulating material. The platinum (Pt) target (Kurt J. Lesker Co.,Clariton, Pa., USA) may include a purity of, e.g., 99.99% or better.Insulating tape 44 may be removed after sputter coating to yield acounter electrode 28 that is physically separated from referenceelectrode 32. Counter electrode 28 and reference electrode 32 may bepositioned at a bottom end of electrochemical flow chamber 2 on top ofthe electrode mounting block (FIG. 1) below the working electrode (FIG.1). In the instant embodiment, counter electrode 28 may includeexemplary dimensions of, e.g., 3 mm (L)×2.3 mm (W). Reference electrode32 may include exemplary dimensions of, e.g., 3 mm (L)×0.5 mm (W). Insome embodiments, counter electrode 28 and reference electrode 32 mayinclude a thickness dimension of about 200 nm, but dimensions are notlimited. Connection wires 40 and 42 may respectively couple counterelectrode 28 and reference electrode 32 to electrochemical workstation(FIG. 1) positioned external to electrochemical device 100. As shown inthe figure, electrochemical flow chamber 2 may include a single inlet 6for introducing samples (e.g., electrolytes, biological fluidsincluding, e.g., cell growth media, and other solutions), and a singleoutlet 8 for removing samples from electrochemical flow chamber 2.

FIG. 2b shows a top perspective view of another exemplary electrodeconfiguration of the present invention of a wire electrode design. Inthe figure, counter electrode 28 and reference electrode 32 may be metalmicrowires constructed of selected metals such as platinum (Pt) or otherconducting metals. Wires 28 and 32 may each be seated in a microchannel(not shown) that fits the dimensions of the wires on the face of theelectrode mounting block (FIG. 1) within electrochemical flow chamber 2.Counter electrode microwire 28 may be separated from reference electrodemicrowire 32. In various embodiments, counter electrode microwire 28 andreference electrode microwire 32 may include an outer diameter of fromabout 25 μm to about 500 μm. In the instant embodiment, connection wires40 and 42 that couple to wire electrodes 28 and 32 may exit through aside of electrochemical flow chamber 2 and connect with anelectrochemical workstation (FIG. 1) located external to electrochemicalflow chamber (FIG. 1). However, insertion location for coupling wires isnot limited. In the instant embodiment, electrochemical flow chamber 2may include a single inlet 6 for introducing samples and a single outlet8 for removing samples from electrochemical flow chamber 2.

FIG. 2c shows a top perspective view of another exemplary electrodeconfiguration of the present invention. In this embodiment, electrodemounting block 20 may be positioned at the bottom of electrochemicalflow chamber 2 below the working electrode (FIG. 1). Counter electrode28 and reference electrode 32 may be sputter-deposited thin filmelectrodes (Denton Vacuum, LLC, Moorestown, N.J.) constructed ofplatinum (Pt) or another suitable electrode material. In someembodiments, counter electrode 28 and reference electrode 32 may includean exemplary thickness of, e.g., 200 nm, but thickness is not limited.Dimensions of counter electrode 28 and reference electrode 32 may betailored to fit dimensions of the electrode mounting block (FIG. 1). Inthe figure, reference electrode 32 may be electrically separate fromcounter electrode 28. In some embodiments, counter electrode 28 may spana surface area on the top of electrode mounting block (FIG. 1) greaterthan about 60%. Reference electrode 32 may occupy a corner of theelectrode mounting block and include a surface area of less than about40%. However, coverage areas are not limited. Connection wires 40 and 42from an electrochemical workstation (FIG. 1) located external toelectrochemical flow chamber 2 may be introduced, e.g., through thebottom of electrochemical flow chamber 2 and coupled, e.g., at thecenter of counter electrode 28 and reference electrode 32, respectively.In the instant embodiment, electrochemical flow chamber 2 may includemultiple (e.g., two or more) inlets 6 for introducing samples anddifferent liquids into flow chamber 2 and multiple (e.g., two or more)outlets 8 for removing samples and liquids from electrochemical flowchamber 2. As shown in the figure, each inlet may be separate fromanother inlet and each outlet may be separate from another outlet.

FIG. 2d shows a top perspective view of another exemplary electrodeconfiguration of the present invention. In the instant embodiment,counter electrode 28 and reference electrode 32 may be thin filmelectrodes constructed of platinum (Pt) or another suitable electrodematerial. Counter electrode 28 and reference electrode 32 may besputter-deposited electrodes (Denton Vacuum, LLC, Moorestown, N.J.).Counter electrode 28 may be separated from reference electrode 32 priorto sputtering with, e.g., a narrow [e.g., 3 mm (L)×0.2 mm (W)] strip ofinsulating tape 44 (Electro Tape Specialities, Inc., FL, USA) or anotherinsulating material. The platinum (Pt) target (Kurt J. Lesker Co.,Clariton, Pa., USA) may include a selected purity of, e.g., 99.99%.Insulating tape 44 may be removed after sputter coating to yield acounter electrode 28 that is physically separated from referenceelectrode 32. Sputter-deposited platinum (Pt) counter electrode 28 andreference electrode 32 may include an exemplary thickness of, e.g., 200nm. Counter electrode 28 may include an exemplary length (L) of about 3mm and an exemplary width (W) of about 2.3 mm, but dimensions are notlimited thereto. Reference electrode 32 may include an exemplary length(L) of about 3 mm width and an exemplary (W) of about 0.5 mm. However,dimensions are not intended to be limited. Connection wires 40 and 42from an electrochemical workstation (FIG. 1) located external toelectrochemical flow chamber 2 may be introduced, e.g., through thebottom of electrochemical flow chamber 2 and coupled, e.g., at thecenter of counter electrode 28 and reference electrode 32, respectively.In the instant embodiment, electrochemical flow chamber 2 may include apatterned inlet 6 with multiple (e.g., three) branches 46 that permitmixing of various samples, liquids, solutions, or battery electrolytesintroduced into flow chamber 2 and a patterned outlet 8 with multiple(e.g., two) branches 46 for retrieving various samples, liquids,solutions, or battery electrolytes from electrochemical flow chamber 2.Number of branches 46 is not limited.

FIG. 3a shows an exemplary working electrode 26 of a single electrodedesign that may be positioned on the underside of silicon nitride (SiN)membrane 16. In the exemplary design, working electrode 26 may becomposed of a thin film of polycrystalline gold (Au) or another suitableelectrode material sputter-deposited onto the underside of SiN supportmembrane 16 using selected coating targets (e.g., Model 30800 seriestargets, Ladd Research Industries Inc., Burlington, Vt., USA). In theinstant design, SiN membrane 16 positioned above working electrode 26may include a length (L) dimension of, e.g., 1.5 mm and a width (W)dimension of, e.g., 1.5 mm (W) for a surface area of 2.25 mm². Herein,the electrode surface area may be presumed to be equal to the geometricarea. In the instant design, working electrode 26 may include a length(L) dimension of, e.g., 2 mm and a width (W) dimension of, e.g., 2 mmfor a total working area of about 4.0 mm². However, areas are notintended to be limited. Working electrode 26 may couple to a conductingmetal connector 48 (e.g., a strip connector), e.g., constructed ofsputter-deposited gold (Au) or another suitable conducting material thatserves as a conductive couple between working electrode 26 and theexternal workstation (FIG. 1).

FIG. 3b shows another electrode configuration of the present inventionof an integrated electrode design. In the instant embodiment, workingelectrode 26 is integrated with a counter electrode 28 and a referenceelectrode 32, with all electrodes positioned beneath the silicon nitride(SiN) support membrane 16. As shown in the figure, electrodes 26, 28,and 32 are all electrically separated. Each electrode 26, 28, and 32 mayfurther couple to respective metal connectors (e.g., a strip connector)48, 50, and 52 constructed of sputter-deposited gold (Au) or anothersuitable metal or conducting material. Strip connectors 48, 50, and 52serve as conductive couples between each electrode and the externalworkstation (FIG. 1).

FIG. 4 illustrates a top perspective view of the assembledelectrochemical device 100. Device 100 includes a working electrode,counter electrode, and reference electrode (not shown) in an integratedelectrode design described previously in reference to FIG. 3b . In theinstant design, working electrode, counter electrode, and referenceelectrode may be sputter-deposited polycrystalline gold films positionedbeneath SiN membrane 16 below top silicon frame 12. Each electrodecouples to an external workstation (FIG. 1) via respective metal wires38, 40, and 42. In the instant embodiment, metal flow tube 54 mayconnect to a fluid inlet (FIG. 1). Metal flow tube 56 may couple to afluid outlet (FIG. 1) within electrochemical device 100. Metal flowtubes 54 and 56 may include an outer diameter of, e.g., 0.025 inches(0.064 mm), an inner diameter of 0.017 inches (0.043 mm), and a lengthof about 0.5 inches (12.7 mm). Metal flow tubes 54 and 56 may furthercouple to flexible flow tubing 62 and 64 (e.g., TEFLON® flow tubing,VICI Valco Instruments, TX, USA) that provide a flow of liquid samplesincluding solutions and electrolytes through flow chamber (FIG. 1).Flexible flow tubing 62 and 64 may include an outer diameter (O.D.) of,e.g., 0.025 inches (0.064 mm), but dimensions are not limited. Topsilicon frame 12, SiN membrane 16, metal coupling tubes 54 and 56 andflexible flow tubing 62 and 64 may be encased in a PDMS enclosure(block) 66. PDMS enclosure 66 may be constructed in a mold (not shown)following assembly of electrochemical flow device 100.

Chemical Imaging

Chemical imaging performed in concert with various probe instrumentsgenerates images of chemical analytes in liquid samples of interest overa selected sampling area. Data provide spectral, spatial, and/ortemporal information across different time and space scales depending onthe various sampling probes used to collect the data. Chemical imagingmay be performed in concert with one or more vacuum-based,surface-sensitive chemical analysis (analytical probe) instrumentsincluding, but not limited to, e.g., X-ray photoelectron spectroscopy(XPS); scanning electron microscopy (SEM); secondary ion massspectrometry (SIMS); time-of-flight secondary ion mass spectrometry(ToF-SIMS); helium ion microscopy (HeIM); Auger electron spectroscopy(AES); Rutherford backscattering spectrometry (RBS); and transmissionelectron microscopy (TEM). The probe instrument analyzes the surface ofthe sample by introducing a probe beam through the detection aperture(FIG. 1) into the liquid sample at a selected depth. For example,surfaces of liquid samples and surfaces at the working electrode/liquidsample interfaces may be probed with probe beams delivered from selectedanalytical instruments at selected depths. In various embodiments, probebeams may probe various depths of liquid samples. In some embodiments,depth may be selected up to about 6 nm that permits selected regions orlayers of the electrode/liquid sample interface to be probed. Forexample, at selected depths, adsorbed molecules, monolayer films (e.g.,2 Å to 10 Å), diffuse-layers (1 nm to 1 μm), and/or modified films(e.g., 1 nm to 1 μm) may be investigated. All depths as will be selectedby those of ordinary skill in the art in view of the disclosure arewithin the scope of the present invention. No limitations are intended.

A liquid sample or interface may be analyzed and data collected at afirst location or surface that provides a first spectral map. Each pixelof the image map may correspond to a m/z spectrum at that pixellocation. The sampling probe may then analyze a second location orsurface of the liquid sample within the sampling area that provides asecond spectral map. The process may be repeated until a preselected,and statistically significant, sampling frequency is obtained. Signalintensities from the collection of mass spectra on the sampling surfacemay be plotted as a function of voltage or scanning depth, which allowsa spatial profile or map to be generated of different chemical speciesidentified within the sampling area for a given sample).

FIG. 5 illustrates an assembled microfluidic electrochemical device(flow cell) 100 mounted to a ToF-SIMS stage 72 of a ToF-SIMS instrument(not shown). ToF-SIMS stage 72 may be configured to insert into a vacuumchamber (FIG. 1) of the ToF-SIMS instrument to provide chemical imaginganalysis of liquid samples introduced into the microfluidic flow chamber(FIG. 1) at the electrode-liquid interface in-situ. In the figure, topframe 12 and SiN support membrane 16 of electrochemical device (flowcell) 100 can be seen. Lateral resolution in the ToF-SIMS instrument maybe from about 20 nm to about 40 nm. Vertical resolution may be about 1nm. In the figure, ToF-SIMS stage 72 may include a pump 74 (e.g., amodel #3000126 electro-osmotic pump, Dolomite, United Kingdom) thatintroduces liquid samples into microfluidic flow chamber (FIG. 1). Pump74 may be enclosed, e.g., in a vacuum enclosure 76 constructed of, e.g.,stainless steel or another suitable material. Fittings 82 that enclosepump 74 within vacuum enclosure 76 may be constructed of a selectedengineering thermoplastic such as polyaryletherketone (PAEK) orpolyether ether ketone (PEEK) that provide suitable mechanical andchemical resistance properties for high-temperature and engineeringapplications. Vacuum enclosure 76 allows pump 74 to operate in thevacuum chamber (FIG. 1) of a selected analytical probe instrument (notshown). Liquid samples may be introduced through flow tubing 62 throughmetal flow tube 54 into microfluidic flow chamber (FIG. 1). Liquidsamples may be removed from the microfluidic flow chamber (FIG. 1)through metal flow tube 56 out through flow tubing 62. Flow tubing 62and 64 (e.g., TEFLON tubing) provides flows of various samplesincluding, but not limited to, e.g., buffer solutions, electrolytes,biological broths, cell media, and other liquid samples delivered intothe flow chamber (FIG. 1). Pump 74 may be powered with a battery 80[e.g., a lithium-thionyl chloride (Li—SOCl₂) battery, Saft America,Valdosta, Ga., USA]. A switch 78 may be used to power pump 74 whenToF-SIMS stage 72 is introduced into the vacuum chamber (FIG. 1) of theToF-SIMS instrument (not shown).

Electrochemical Analysis

Electrochemical analysis may be provided by the present inventionindividually or simultaneously with chemical imaging analyses, asdescribed above. An electrochemical workstation (FIG. 1) may be used todeliver potentials to electrodes (FIG. 1) of the microfluidicelectrochemical device. FIG. 6 shows a typical cyclic voltammogram (CV)curve obtained from cyclic voltammetric (electrochemical) analyses. TheCV curve obtained in high vacuum plots current (e.g., in μAmps) againstpotential (in Volts). Here, data were collected using a 10 mM KIelectrolyte solution as a liquid sample at scan voltages ranging from−0.2 V to 0.9 V at a scan rate of 100 mV/s as measured by the gold (Au)working electrode (FIG. 3a ). The CV curve collected at high vacuum hasthe same features as the CV curve obtained at ambient conditions.Results demonstrate the ability of the microfluidic electrochemicaldevice to provide both electrochemical analysis and chemical imaging ofchemical species (i.e., chemical speciation) positioned at selecteddepths and layers at surfaces of liquids, other flowable samples, and atthe solid electrode-liquid interface. A slight potential shift in the CVcurve may be due to differences in the selection of the referenceelectrode that do not affect performance of the microfluidicelectrochemical device. Thus, no limitations are intended.

FIGS. 7a-7d show representative ToF-SIMS 2D images of the chemicalspecies IO₃ ⁻, I₂ ⁻, I₃ ⁻ and AuI₂ ⁻ acquired at +0.8 V with themicrofluidic electrochemical device of the present invention. ToF-SIMSis a representative instrument suitable for chemical imaging. Each pixelin the 2D images includes data corresponding to a spatial distributionof chemicals (including, e.g., transient species and chemical products)identified at the selected location or distance from the workingelectrode at the selected potential. Chemicals identified at theselected locations may be reflected in the m/z spectrum (see, e.g.,discussion in reference to FIG. 8). Here, the working electrode wasconstructed of polycrystalline gold (Au) to allow direct electrochemicalmeasurements at the electrode-solution interface. In the electrochemicalprocess, electrons are removed from the metallic gold, and subsequentgold ions form strong complexes with iodide in the electrolyte (or goldiodide adlayers on the electrode) according to reaction [1]:

Au⁺ _((aq))+2I⁻ _((aq))

AuI₂ ⁻ _((aq))  [1]

Reaction [1] is followed by four electrode reactions considered to bedirectly related to the total amount of chemisorbed iodine includingsolution redox, anodic oxidation of chemisorbed iodine, and iodatereduction as shown in reactions [2]-[5], respectively:

½I_(2(aq)) +e ⁻

I_((aq))  [2]

I_(2(aq))+I⁻ _((aq))

I₃ ⁻ _((aq))  [3]

I_((ads))+3H₂O

IO₃ ⁻ _((aq))+6H⁺ _((aq))+5e ⁻  [4]

IO₃ ⁻ _((aq))+6H⁺ _((aq))+5e ⁻

½I_(2(aq))+3H₂O  [5]

FIG. 8 presents ToF-SIMS m/z (chemical imaging) spectra that plot thenormalized intensity as a function of the electrochemical (CV analysis)potential (from −0.2 V to 0.8 V, respectively) measured with a gold (Au)working electrode (FIG. 3a ) in a supporting KI electrolyte. Iodineadlayers on surfaces of single crystal gold electrodes are known toexhibit various potential-dependent phase transitions that affect theunderpotential deposition of metal ions on the electrode surfaces.“Underpotential Deposition (or UPD)” refers to a phenomenon in whichmetal cations are reduced to their solid metal form and areelectrodeposited to the electrode surface at a potential that is lessnegative (and thus more electrochemically favored) than should normallyoccur at normal (or Nernst equilibrium) conditions. The observance ofUPD is attributed to interactions between the metal ions to beelectrodeposited and the metal on the electrode surface.

In the figure, only m/z peaks of interest are presented for clarity.Features of the CV are in agreement with reported literature values.Normalized ToF-SIMS m/z spectra obtained at different potentials clearlyillustrate for the first time the molecular composition of the redoxreaction products and intermediate species at different stages of theredox cycle. At −0.2 V, no redox reaction occurs. Thus, only I⁻ isobserved. The same is true at 0 V. Areas I and II (−0.2 V to +0.2 V)show formation of AuI₂ ⁻, I₂ ⁻, and I₃ ⁻, respectively, which contrastswith reported literature results which indicate that no redox reactionoccurs there. At +0.2 V, the complex AuI₂ ⁻ as well as I₂ ⁻ and I₃ ⁻ areobserved, indicating that reactions [1]-[3] occur in this region. AreaIII (i.e., +0.2 to +0.4 V) may reflect various equilibria speciesincluding, e.g., I⁻, I₂, and I₃ ⁻ corresponding to reactions [2] and[3]. At +0.4 V, no I₃ ⁻ is observed, which is attributed to a redoxreaction occurring at this voltage that favors dissociation of I₃ ⁻.Area IV (i.e., +0.4 to +0.6 V) reflects oxidation of I₂ to IO₃ ⁻ asgiven by reactions [2]-[4]. Area V (i.e., +0.6 to +0.8 V) reflectsreduction of IO₃ ⁻ to I₂ ⁻, respectively. At +0.8 V, the expectation isthat IO₃ ⁻ can form. However, the ToF-SIMS m/z spectrum corresponding tothe CV results indicates that IO₃ ⁻ can form at lower potentials.

Applications

Microfluidic electrochemical devices of the present invention providesimultaneous chemical imaging and electrochemical analyses of chemicalentities at the electrode-liquid (e.g., electrolyte) interface in-situ.In some embodiments, electrochemical devices of the present inventionare portable, multimodal lab-on-a-chip devices. The present inventioncan, for the first time, provide combined electrochemical analyses andchemical imaging of electrode-liquid (or other solid-liquid) interfacesin concert with surface-sensitive vacuum techniques such as ToF-SIMSunder vacuum conditions in-situ.

In various embodiments, the present invention has many varied andpotential applications for study of a wide variety of analytes in liquidsamples. The present invention provides measurement, determination,and/or characterization of various analytes including, but not limitedto, e.g., redox-active analytes; biological analytes including, e.g.,cells and bacteria; cell growth media; biofilms; chemical analytes;molecular analytes; solid analytes; mixtures; nanoparticles; complexanalytes; transient analytes; battery electrolytes; buffer solutions;including combinations of these various analytes.

In some embodiments, electrode-electrolyte interactions in fluid motionin real time may be analyzed. Moreover, distributions of intermediatespecies in electrode-electrolyte reactions may be discerned usingchemical imaging in real-time as reactions occur and potential-dependentsurface changes may be followed in real-time in-situ. The followingexamples provide a further understanding of various aspects of theinvention.

Example 1 Microfluidic Electrochemical Cell

The microfluidic electrochemical device was fabricated using a softlithography approach. Exemplary dimensions of the electrochemical flowchamber were approximately 2.5 mm×2.5 mm×0.3 mm. The photomask wasdesigned using AutoCAD software and printed with a mask printer(Intelligent Micro Patterning LLC, Model SF-100 Xpress). A template forcasting the electrochemical flow chamber was made with SU-8 photoresist(Microchem, Newton, Mass.) on a silicon substrate. The template includeda flow channel with a depth of 300 μm equal to the distance between theworking electrode and a counter electrode. A 10:1 ratio (w/w) ofpolydimethylsiloxane (PDMS) prepolymer and curing agent (Sylgard 184,Dow Corning Co., Midland, Mich., USA) were thoroughly mixed, degassedunder vacuum, poured onto the patterned template to a thickness of 1 cm,and cured in an oven at 75° C. overnight.

Example 2 Preparation of Potassium Iodide Sample Solutions

Aqueous 10 mM solutions of potassium iodide (Aldrich, 99.9%) wereprepared in ultrapure water (18.2 MΩ·cm) obtained with a waterpurification system (Milli-Q Integral Water Purification System, EMDMillipore, Billerica, Mass., USA) at 25° C. Oxygen in the solutions wasdepleted using a nitrogen bubbler before filling the microfluidicelectrochemical device. Solutions were degassed using a commercialdegasser (e.g., a DU series 200 μL Degasys Ultimate internal volumedegasser, Sanwa Tsusho Co., Ltd., Tokyo, Japan) to limit pressurebuild-up and bubble formation inside the device. The microfluidicelectrochemical device was filled with solution using a syringe pump(Harvard apparatus, Holliston, Mass., USA).

Example 3 Operation in ToF-SIMS

The vacuum compatible microfluidic electrochemical device was deployedinto a vacuum chamber of a commercial ToF-SIMS instrument (IONTOF GmbH,Münster, Germany). The microfluidic electrochemical device was checkedfor leaks in a vacuum chamber before usage. Vacuum pressure in the flowchamber of the electrochemical device during measurements was 2.0×10⁻Torr to 5.5×10⁻⁷ Torr. The ToF-SIMS instrument was configured to rastera focused (0.2 μm) primary ion beam through the SiN window of theelectrochemical device over sample liquids. Ejected secondary ions weremass-analyzed to form surface chemical maps with a lateral resolution ofabout 0.2 μm. ToF-SIMS measurements were performed at a beam current of˜1.0 pA with a beam width of 125 ns and a repeated frequency of 16.7kHz. ToF-SIMS provides molecular information, e.g., on the top layer(e.g., ˜6 nm) of sample liquids and other solutions. ToF-SIMS mayemploy, e.g., 25 keV Bi⁺ ions as probes. Ions were detected at fromabout 0 eV to about 10 eV. In the imaging mode, an area of 2×2 μm² wasselected. The Bi⁺ beam was scanned with 128×128 pixels with a totalintegration time of 295 seconds.

Example 4 Electrolyte Solutions

In exemplary tests, electrolyte solutions were used to demonstratereal-time observation of chemical species formed as a result of redoxreactions in the microfluidic electrochemical flow device, e.g., thegold iodide adlayer at the surface of the electrolyte in contact withthe working electrode in-situ. Electrolytes were simultaneously analyzedusing cyclic voltammetry (CV) and Time-of-Flight Secondary Ion MassSpectrometry (e.g., ToF-SIMS). Potential-dependent changes at theelectrode surface and the electrolyte composition are characterized byToF-SIMS imaging that reflect progression of electrochemical redoxreactions as a function of time. Electrochemical results and ToF-SIMSm/z spectra obtained at different potentials show for the first time thegold adlayer and transient species formed during charge-transferprocesses in-situ, as detailed herein.

Example 5 Cyclic Voltammograms

Cyclic voltammograms (CVs) were collected using an electrical-chemicalstation (Model 824 Electrochemical Detector, CH Instruments, Inc.,Austin, Tex., USA). The reference electrode was a 200 nm Pt thin film.Voltammograms were collected at ambient and high vacuum (<5×10⁻⁷ Torr)conditions in an electrolyte at 25° C. performed at a scan rate of 20mV/s. The scan was initiated at −0.2V. Potential was advanced at a stepof 0.2V from −0.2 V to 0.9 V. Each voltage was held for a selectednon-limiting time. ToF-SIMS data were also acquired. FIG. 6 shows atypical cyclic voltammogram obtained in concert with the presentinvention as a function of applied voltage. FIG. 8 shows representativeToF-SIMS m/z spectra acquired in-situ at various voltages in concertwith the present invention.

Example 6 Analysis of Battery Electrolytes

The microfluidic electrochemical device of FIG. 1 may be used with aworking electrode constructed of a sputter-deposited metal or metaloxide positioned on the backside of the SiN membrane. An (e.g., 1M)electrolyte may be injected into the electrochemical device via asyringe pump. An electrochemical station may be coupled to themicrofluidic electrochemical device to apply various potentials betweenthe working electrode (e.g., gold electrode) and counter electrodewithin the microfluidic electrochemical device to induce electrontransfer within the electrolyte in the flow chamber. Electrolytes may beanalyzed in either static or dynamic flow mode. During electrochemicalanalysis, a ToF-SIMS or other surface-sensitive analytical instrumentmay be used to simultaneously probe the surface of the electrode andelectrolyte interface. Results similar to FIG. 6 and FIG. 8 may beobtained. The m/z data and charge/discharge curves acquired as afunction of time and potential may be used to provide chemical imagingof chemical species such as complexes formed from charge transferreactions at electrode and electrolyte surfaces that characterize theelectrolyte and elucidate battery performance at the molecular scale,e.g., for battery applications that enables design of better electrodematerials.

Example 7 Analysis of Biofilms

FIG. 9 shows microfluidic device 100 of the present invention configuredas a growth reactor for growth and analysis of biofilms. Biologicalbroths may also be analyzed. In the figure, microfluidic device(reactor) 100 includes a flow chamber 2 configured with a top frame 12(e.g., a silicon frame), a support membrane 16 (e.g., a SiN supportmembrane), a detection aperture 18, and a flow channel 4 through flowchamber 2 that connects to an inlet channel (inlet) 6 and outlet channel(outlet) 8. Flow chamber 2 may be constructed of, e.g., a PDMS elastomeras described previously herein in reference to FIG. 1. Fluid inlet 6into reactor 100 and fluid outlet 8 out of reactor 100 was connected toPFTE tubing (FIG. 4 and FIG. 5) to provide a fluid flow path throughmicrofluidic flow chamber 2. A needle was inserted into the inlettubing, and reactor 100 was coupled to a custom-made manifold (notshown) that allowed delivery of the sample medium and other solutionsinto and out of reactor 100. A 70% ethanol solution was flowed throughflow chamber 2 for three hours to sanitize microfluidic flow channel 4and flow chamber 2. A minimum of five volume changes of filtered water(>18.2 MS)) was then flowed to flush the ethanol solution from flowchamber 2. Two syringes (not shown) were used as fluid reservoirs toprovide a sufficient quantity and flow of growth medium through reactor100 to grow biofilm 84 without changing the supply of growth medium andother solutions. The manifold was connected to microfluidic reactor 100via the needle. A sterile growth medium was flowed through flow chamber4 overnight prior to inoculating reactor 100.

Biofilm growth may follow biofilm growth guidelines detailed, e.g., byMcLean et al. in J. Microbiol Methods 74: 47-56). A late-log phasebacterial culture was harvested by centrifugation (5000×g, 10 minutes)and resuspended in an equal volume of sterile medium. One milliliter (1mL) of the resuspended bacterial culture medium was flown throughmicrofluidic reactor 100 via syringe (not shown) to inoculate reactor100. The syringe was then exchanged with two syringes containing sterilegrowth medium. Flow mode was used during growth of biofilm 84. Cellgrowth medium was flowed at room temperature through microfluidicreactor 100 for five to six days at a flow rate permissive for sub-oxicbacterial growth to form a biofilm 84 on the underside of supportmembrane 16 below detection aperture 18. Biofilm 84 growth was monitoredin real-time using a confocal microscope or other light microscope.Biofilm 84 achieved an average depth of 300 μm and a non-limiting widthand length dimension of 500 μm. Static mode may be employed after growthof the biofilm 84 during chemical imaging. After growth of the biofilm84, microfluidic reactor 100 containing the hydrated biofilm 84 wasassembled onto a portable ToF-SIMS stage (FIG. 5) and introduced intothe vacuum chamber (FIG. 1) of a ToF-SIMS instrument (or otheranalytical instruments) for chemical imaging in-situ. ToF-SIMS has anadvantage in that it provides molecular recognition for biological (andother organic) molecules. Peaks (m/z) representing quasi molecular ionsformed from loss of a hydrogen (e.g., [M-H]⁻) are commonly observed inthe ToF-SIMS spectra in negative mode. Electrodes described in referenceto FIG. 1 may also be introduced into flow chamber 2 to provide combinedelectrochemical analysis and chemical imaging of biofilms 84 grown inmicrofluidic reactor 100, as well as cell media or biological brothmedia. Biofilm 84 and the cell medium (not shown) were imaged using aprimary probe beam 22. Mass spectrometry ion data were collected with asecondary ion beam 24.

FIG. 10a presents a negative ToF-SIMS m/z spectrum showingcharacteristic fragment peaks for a dry Shewanella biofilm sample placedon a clean silicon wafer. FIG. 10b presents a negative ToF-SIMS m/zspectrum showing characteristic fragment peaks for the hydratedShewanella biofilm grown in the microfluidic reactor chamber in-situ.The ToF-SIMS spectrum shows characteristic m/z peaks for selected fattyacids fragments including, e.g., C₁₄ fatty acid fragments (m/z of 227)and C₁₅ fatty acid fragments (m/z 241). Differences are observed betweenthe liquid sample as compared to the dry sample. For example, additionalpeaks are observed for the liquid sample in the m/z range between 200amu and 300 amu. FIG. 10c shows a ToF-SIMS m/z spectrum collected forthe growth medium in the microfluidic channel acquired as a control.TABLE 1 lists peak (m/z) assignments and identities of selected fattyacid fragments observed in FIGS. 10a-10b .

TABLE 1 compares peak (m/z) assignments for fatty acid fragmentsidentified from ToF-SIMS chemical imaging analyses of biofilm samplesacquired with the microfluidic reactor of the present invention withtheoretical peak assignments obtained from the LIPID MAPS StructureDatabase (LMSD). Mass Peak LMSD Name Species Formula (m/z) (m/z) Lauricacid [M − H]− C₁₂H₂₃O₂ ⁻ 199.14 199.17 Tridecylic acid [M − H]− C₁₃H₂₅O₂⁻ 213.15 213.19 Myristic acid [M − H]− C₁₄H₂₇O₂ ⁻ 227.16 227.20Pentadecylic acid [M − H]− C₁₅H₂₉O₂ ⁻ 241.18 241.22 Palmitic acid [M −H]− C₁₆H₃₁O₂ ⁻ 255.18 255.23

Characteristic peaks at m/z values of 199, 213, 227, 241, and 255 werefound in the dry biofilm sample corresponding to various fatty acidfragments. The peak at an m/z value of 199 was attributed to C₁₂H₂₃O₂ ⁻(Lauric acid). The peak at an m/z value of 213 was attributed toC₁₃H₂₅O₂ ⁻ (Tridecylic acid). The peak at an m/z value 227 wasattributed to C₁₄H₂₇O₂ ⁻ (Myristic acid). The peak at an m/z value 241was attributed to C₁₅H₂₉O₂ ⁻ (Pentadecylic acid). The peak at an m/zvalue 255 was attributed to C₁₆H₃₁O₂ ⁻ (Palm itic acid). Slight shiftsin m/z values for fatty acid fragments reported here when compared topeaks in the LIPID MAPS Structure Database (LMSD) [www.lipidmaps.org]may be attributed to instrument systematic differences and the differentstrains of bacteria analyzed. In FIG. 10b , the hydrated (wet) biofilmsample shows more peaks when compared to the dry biofilm sample. Slightm/z shifts are attributed to instrument responses for a givenexperiment. For example, the 199 mass peak for lauric acid ([C₁₂H₂₃O₂]⁻)is observed at 199.14 amu, which is consistent with the theoreticalvalue (199.17 amu). And, the 213 mass peak for tridecylic acid([C₁₃H₂₅O₂]⁻) is observed at 213.15 amu, in good agreement with itstheoretical value (213.19 amu). In the control experiment (FIG. 10c ),no discernible peaks were found, which demonstrates that peaks observedfor the dry biofilm sample and the wet biofilm sample stem are due tothe biofilm. Resolution and intensity of peaks in the liquid biofilmsample may be increased by optimization of analysis parameters.

FIG. 11a shows ToF-SIMS depth profiles of key components in the hydratedbiofilm sample. FIG. 11b shows principle component analysis (PCA)results that compare distinctions between the hydrated biofilm, the cellgrowth medium, the dry biofilm sample, and the SiN membrane of themicrofluidic device. FIG. 11c shows 2D chemical images of fragmentsobtained from the biofilm and for the growth medium at an m/z 227 (C₁₄fatty acid) and fragments at an m/z of 241 (C₁₅ fatty acid),respectively.

Example 8 Buffer Solution Analysis

The microfluidic electrochemical device of FIG. 1 was used. Threeprotein-modified gold nanoparticle containing solutions (SPI SuppliesLLC, West Chester, Pa., USA) were tested. Proteins were goat anti-mouseIgG (H+L), goat anti-rabbit IgG, and Strepavidin, respectively. Goldnanoparticles had a mean diameter of, e.g., −5 nm. Nanoparticles weresuspended in 20 mM Tris/NaCl buffer, and diluted with deionized water toa concentration of 4 μg/mL. Solutions were introduced into themicrofluidic device with a syringe pump (Harvard apparatus, Holliston,Mass.). Aqueous solutions were degassed by a commercial vacuum degasser(Chrom Tech, Inc., MN, USA). Wet nanoparticle samples were prepared bydepositing a drop of a solution on a clean silicon wafer [e.g., aSi(100) wafer, University Wafer, Boston, Mass., USA]. Dry samples wereprepared by drying a wet sample deposited on a silicon substrate (e.g.,clean silicon wafers) under ambient conditions. Vacuum pressure in themicrofluidic flow chamber was between about 2×10⁻⁷ mbar to about 4×10⁻⁷mbar. Pressure increased during measurements to between about 3×10⁻⁷mbar and about 5×10⁻⁷ mbar. The narrow pressure range indicates that norelease, spraying, or spreading of aqueous solutions occurs through thedetection aperture. Both SEM and ToF-SIMS instruments were used to studywet and dry samples in the microfluidic device. Elemental compositionfrom SEM and molecular identification of these samples by ToF-SIMS wereobtained and compared.

While preferred embodiments of the present invention have been shown anddescribed, it will be apparent to those of ordinary skill in the artthat many changes and modifications may be made without departing fromthe invention in its true scope and broader aspects. Appended claims aretherefore intended to cover all such changes and modifications as fallwithin the spirit and scope of the present invention.

1-24. (canceled)
 25. An electrochemical device for combinedelectrochemical analysis and chemical imaging of analytes at anelectrode-liquid sample interface in situ under vacuum, the devicecomprising: an electrochemical microfluidic flow cell defining a flowchamber; a support membrane within the cell, the membrane defining atleast one detection aperture, the aperture providing an opening throughwhich probe beams can be delivered to the interior of the flow chamber;and a pair of working electrodes within the chamber of the cell.